The invention relates to regulation of the complement system employing modified proteins of the gene cluster which encodes regulators of complement activation (RCA). More specifically, it concerns analogs of proteins of this system which have modified binding specificities and affinities.
The complement system serves to aid in the removal of foreign substances and of immune complexes from animal hosts. A recent summary of the nature of this system and its regulation was published by Hourcade, D., et al., Advances in Immunol (1989) 45:381-416, which is incorporated herein by reference.
Briefly, as set forth in this review, the complement system generates, either by a xe2x80x9cclassical pathwayxe2x80x9d or an xe2x80x9calternative pathway,xe2x80x9d the important protein C3b which binds to target immune complexes or foreign substances and marks them for destruction or clearance. C3b is generated from its precursor C3 by the proteolytic enzymes collectively designated xe2x80x9cC3 convertase.xe2x80x9d One form of C3 convertase is generated in the classical pathway by the association of the proteins C4b and C2a. The other form is generated in the alternative pathway by association of C3b and Bb. Both C3 convertases can associate with an additional C3b subunit to form the C5 convertases, C3bBbC3b and C4bC2aC3b, both of which are active in the production of the C5-C9 membrane attack complex which can cause cell lysis, and in the production of C5a, a major pro-inflammatory agent. Thus, both C3b, and less directly, C4b, are agonists in the complement system.
FIG. 1 shows these relationships.
The complement system is a blessing when it operates as intended, but it has to be kept under control because it can mark the body""s own tissues for destruction. Thus, humans and other animals provide mechanisms whereby various components of the complement system which behave in this agonistic fashion can be destroyed, especially when associated with cells which are endogenous to the host.
There are two general mechanisms for this inhibition of the effects of the complement system. The first mechanism is generally reversible, facilitating the dissociation of the C3 convertasesxe2x80x94i.e., C3b from Bb and C4b from C2a. Facilitation of dissociation is sometimes known as decay acceleration. The dissociation may also involve reversible binding of the antagonist proteins to C3b or C4b components, thus preventing their reassociation. The other mechanism, which is an irreversible inactivation process, results from proteolytic cleavage of the C3 convertase components C3b or C4b by the serine protease factor I. This proteolytic cleavage occurs only in the presence of a cofactor. Both general regulatory mechanisms, the facilitation of dissociation of C3b and C4b and the inactivation of C3b and C4b through cleavage by factor I, also apply to the inhibition of the alternative pathway C5 convertase (C3bBbC3b) and the classical pathway C5 convertase (C4bC2aC3b).
The proteins encoded by a region of the genome which is designated the xe2x80x9cregulators of complement activationxe2x80x9d (RCA) gene cluster are involved in both of the foregoing mechanisms. Currently, it is known that at least six proteins are encoded by this region. These are summarized in Table 1.
These proteins share certain structural similarities which will be further described below.
The reversible binding to C4b or C3b to dissociate the C3 convertases is effected by two plasma proteins designated C4 binding protein (C4bp) and factor H, and by two membrane proteins designated decay acceleration factor (DAF) and complement receptor 1 (CR1). Reversible binding to C4b is effected by C4bp, DAF and CR1 while reversible binding to C3b is effected by factor H, DAF and CR1.
The irreversible inactivation of the C3 convertases resulting from proteolytic cleavage of convertase components C3b or C4b by the enzyme factor I can occur by virtue of cofactor activity effected by the above-mentioned factor H and C4bp in the plasma and by CR1 and membrane cofactor protein (MCP) at the cell surface. Cofactor activity for cleavage of C3b is effected by factor H, CR1 and MCP while cofactor activity for cleavage of C4b is effected by C4bp, CR1 and MCP. It is also possible that the sixth protein, complement receptor 2 (CR2), has this cofactor activity at the cell surface.
Thus, for the six proteins encoded by the RCA gene cluster, factor H, C4bp, and CR1 have both reversible dissociation activity and irreversible cofactor activity; DAF has only reversible dissociation activity, and MCP and possibly CR2 have only irreversible cofactor activities. CR1, DAF and MCP interact with both C3b and C4b; C4bp interacts primarily with C4b, and factor H interacts primarily with C3b.
The Hourcade article referenced and incorporated above describes the relationship of these proteins as judged by comparison of deduced amino acid sequences from cDNAs. The cDNAs corresponding to CR1, CR2, DAF, MCP, C4bp, and factor H have all been obtained and sequenced. Evaluation of these comparative sequences has lead to the alignment set forth in FIG. 2 which shows the organization of the RCA proteins into SCR-containing and non-SCR-containing regions with the N-terminal ends at the left. In this figure, TM refers to transmembrane domain, C to cytoplasmic domain, O to O-linked glycosylation domain, G to glycolipid anchor, U to domain with unknown significance and D to a disulfide bridge-containing domain.
It is seen that there is considerable uniformity across the RCA family of proteins. All of them are composed of 60-70 amino acid repeating units commonly designated xe2x80x9cshort consensus repeatsxe2x80x9d (SCRs). Each SCR shares a number of invariant or highly conserved amino acid residues with other SCRs in the same protein or SCRs in other family members. Those members of the family which are membrane bound also have, at their C-termini, transmembrane regions and intracellular regions or else they have a glycolipid anchor.
The SCRs form the extracellular portions of those members of the family which are membrane-bound and almost all of the protein structure in the secreted members. Two covalently-crosslinked cysteine pairs establish two loops within each SCR. The smallest family members are DAF and MCP; each contains 4 SCRs followed by an O-linked glycosylation region. DAF is terminated with a glycolipid anchor while MCP ends with an extracytoplasmic segment of unknown significance, a transmembrane region and an intracellular domain. Of the secreted members of the family, factor H contains 20 SCRS, while the native form of C4bp is an association of seven subunits of 8 SCRs (the C4bp alpha chains) and one subunit of 3 SCRs (the C4bp beta chain). Both C4bp chains conclude with non-SCR domains that interconnect the chains through disulfide linkages. CR2 contains 16 SCRs, a transmembrane region and an intracellular domain. CR1 contains 4 repeating units of 7 similar SCRs (long homologous repeats or LHRs) numbered 1-28 followed by an additional 2 SCRs designated 29 and 30, a transmembrane region and an intracellular region.
Klickstein, L. B., et al., J Exp Med (1988) 168:1699-1717, described the identification of distinct C3b and C4b recognition sites in CR1 using deletion mutagenesis. They concluded that a single primary C4b binding site is located in SCR 1-2, while two major C3b binding sites are located in SCR 8-9 and SCR 15-16. C3b cofactor activity was localized to SCR 8-9 and SCR 15-16.
Hourcade, D., et al., J Exp Med (1988) 168:1255-1270, described a cDNA clone designated CR1-4 that encodes the first 8 and one-half amino terminal SCRs of CR1. This cDNA was transfected into COS cells and this resulted in the synthesis of a secreted truncated form of CR1 with a molecular weight of 78 kd (Krych et al., 1991). This shortened form of the protein, as shown hereinbelow, binds mainly C4b.
The multiple binding sites of CR1 can cooperate in their interactions with C3b-containing targets. In vitro, CR1 binds C3b-C3b dimers much more tightly than C3b monomers because binding to dimers can occur simultaneously at two sites in the same CR1 molecule (Wong and Farrell J Immunol (1991) 146:656; Ross and Medof Adv Immunol (1985) 37:217). Deletion of one of the two primary C3b binding sites can reduce the binding of CR1 to C3b-C3b by a factor of 10 (Wong and Farrell J Immunol (1991) 146:656.) It is likely that the primary C4b binding site also cooperates with the primary C3b-binding sites in interactions with targets that contain both C3b and C4b. These effects have an important consequence in vivo: CR1 has a higher affinity for targets densely coated with C3b and with targets densely coated with C3b plus C4b.
In addition, the C5 convertases, which are important in the stimulation of inflammation and in lysis of some target cells, are composed of multiple CR1 ligands: The classical C5 convertase contains C3b and C4b (C4bC3bC2a) while the alternative pathway C5 convertase contains two C3b proteins (C3bC3bBb). Inactivation of the C5 convertases by CR1 can also involve cooperation between more than one CR1 binding site. Indeed, it has been shown (Wong and Farrell J Immunol (1991) 146:656) that more than one CR1 C3b binding site may be essential for effective inhibition of alternative pathway C3 and C5 convertases.
It is recognized that the proteins encoded by the RCA gene cluster could be prepared recombinantly and used in diagnosis and therapy for the regulation of the complement system. The problems of transplantation of xenografts were reviewed by Platt, J. L., et al., in Immunology Today (1990) 11:450-457. Evidence has accumulated that the immediate hyperacute rejection of discordant xenografts is caused by recipient complement activity. Transgenic animals expressing human complement regulators (such as DAF or MCP) on cell surfaces could be an abundant source of organs that would be protected form hyperacute rejection in human recipients. A soluble complement inhibitor could also play a role in protecting xenografts from complement-mediated rejection.
The ability of a recombinant soluble form of CR1 to inhibit inflammation in the reversed passive Arthus reaction in rats was described by Yeh, C. G., et al., J Immunol (1991) 146:250-256. This soluble CR1 was obtained in Chinese hamster ovary (CHO) cells from expression of a CR1 genetic construct which had been mutated to remove the transmembrane and cytoplasmic domains. The ability of a similar soluble CR1, produced also recombinantly in CHO cells, to inhibit post-ischemic myocardial inflammation and necrosis in rats was reported by Weissman, H. F., et al., Science (1990) 249:146-151.
Proteins related to the RCA proteins have also been shown to be produced by viruses, presumably as a mechanism whereby infection by the virus can be facilitated (Kotwaal, J., et al., Nature (1988) 335:176-178; McNearney, T. A., J Exp Med (1987) 166:1525-1535).
Complete inhibition of the complement system on a long-term basis is not likely to be desirable in most individuals. In some cases of autoimmune disease, inhibition of the classical pathway alone may be sufficient. In the case of the xenograft, however, stringent inhibition of both pathways may be important. Similar stringency may be required for other applications. Accordingly, alternative modulators of the complement system with regulatable binding activities would be desirable. The present invention provides a means to prepare modified forms of RCA-encoded proteins with altered binding specificities and affinities which permit closely controlled modulation of the complement system.
The invention is directed to analogs of RCA-encoded proteins which analogs are modified full-length or truncated forms of these regulatory proteins. These analogs also include modifications of RCA hybrids, which can contain one or more SCRs from more than one RCA protein, and modifications of RCA recombinants in which SCRs are arranged in new orders. The modifications reside in the adjustment of the binding specificity and affinity of the analog as compared to the protein including the truncated, hybrid or recombined form thereof from which the modification was taken. These analogs are useful in controlling the complement system, and thus may be useful in the treatment of autoimmune diseases, the suppression of rejection of transplants, in diagnosis and the reduction in tissue damage associated with myocardial infarctions and cerebral vascular accidents. They may also play a role in the diagnosis of conditions associated with complement activation and immune complex formation.
Thus, in one aspect, the invention is directed to analogs of RCA-encoded proteins including their truncated, hybrid, and recombined forms, which analogs have different binding specificities and/or affinities from the unmodified protein. In another aspect, the invention is directed to recombinant materials useful in the production of these analogs. In still another aspect, the invention is directed to pharmaceutical compositions wherein these analogs are active ingredients and to methods of diagnosis using these analogs. In still another aspect the invention is directed to methods of preparing the analogs of the invention.